ELOVL5 Antibody

What is ELOVL5 and why is it important in research?

ELOVL5 (Elongation of Very Long Chain Fatty Acids protein 5) is a condensing enzyme that catalyzes the first and rate-limiting reaction in the long-chain fatty acid elongation cycle. It functions specifically toward polyunsaturated acyl-CoA with higher activity toward C18:3(n-6) acyl-CoA, playing a crucial role in elongating fatty acid substrates ranging from 16 to 20 carbons . ELOVL5 is critical for maintaining cellular membrane integrity and signaling pathways. Recent research has demonstrated its involvement in various pathological conditions, including cancer metastasis, metabolic disorders, and neurological diseases, making it an important target for antibody-based research .

What tissue expression pattern does ELOVL5 exhibit?

ELOVL5 is predominantly expressed in the adrenal gland and testis, but significant expression is also found in the lung, brain, prostate, and liver tissues . Interestingly, ELOVL5 is localized to the sebaceous glands in pheromone-producing regions of the skin, suggesting potential roles in pheromone production and regulation . At the subcellular level, ELOVL5 is primarily localized in the endoplasmic reticulum as a multi-pass membrane protein, where it functions in the fatty acid elongation process .

What types of ELOVL5 antibodies are currently available for research?

Several types of ELOVL5 antibodies are available with varying characteristics:

These antibodies are available in various conjugated forms including HRP, PE, FITC, and Alexa Fluor conjugates for specialized applications .

How should I optimize western blotting conditions for ELOVL5 detection?

For optimal western blotting results with ELOVL5 antibodies, consider the following protocol:

• Sample preparation: Use RIPA buffer with protease inhibitors to extract ELOVL5 from tissues or cells.

• Protein loading: Load 20-50 μg of total protein per lane.

• Gel percentage: Use 10-12% SDS-PAGE gels for optimal separation.

• Transfer conditions: Transfer at 100V for 60-90 minutes using PVDF membranes (preferred over nitrocellulose).

• Blocking: 5% non-fat milk in TBST for 1 hour at room temperature.

• Primary antibody dilution: Typically 1:1000-1:6000 depending on the specific antibody .

• Incubation: Overnight at 4°C with gentle rocking.

• Detection: ELOVL5 is observed at approximately 25-35 kDa, with some antibodies detecting it around 25 kDa despite a calculated molecular weight of 35 kDa .

Validated positive controls include HeLa cells, MCF-7 cells, mouse kidney tissue, and rat kidney tissue .

What are the recommended protocols for immunohistochemistry with ELOVL5 antibodies?

For successful IHC using ELOVL5 antibodies:

• Fixation: 10% neutral buffered formalin, paraffin-embedded sections.

• Section thickness: 4-6 μm.

• Antigen retrieval: TE buffer pH 9.0 is recommended, though citrate buffer pH 6.0 can be used as an alternative .

• Antibody dilution: 1:50-1:500 for rabbit polyclonal antibodies; 1:2500-1:5000 for certain antibodies .

• Incubation: 1-2 hours at room temperature or overnight at 4°C.

• Detection system: HRP/DAB or fluorescent secondary antibodies.

• Counterstain: Hematoxylin for brightfield or DAPI for fluorescence.

• Positive controls: Mouse liver tissue has been validated for ELOVL5 IHC .

How should I approach immunofluorescence experiments using ELOVL5 antibodies?

For immunofluorescence studies:

• Cell preparation: Fix cells with 4% paraformaldehyde for 15 minutes at room temperature.

• Permeabilization: 0.1-0.25% Triton X-100 in PBS for 10 minutes.

• Blocking: 1-5% BSA or 5-10% normal serum in PBS for 30-60 minutes.

• Primary antibody: Dilute ELOVL5 antibodies at 1:50-1:500 .

• Incubation: 1-2 hours at room temperature or overnight at 4°C.

• Secondary antibody: Fluorophore-conjugated antibodies (Alexa Fluor dyes recommended).

• Counterstaining: DAPI for nuclear visualization.

• Mounting: Anti-fade mounting medium to prevent photobleaching.

HepG2 and HeLa cells have been confirmed as suitable positive controls for ELOVL5 immunofluorescence studies .

How do I select the appropriate ELOVL5 antibody for studying its role in fatty acid metabolism?

Selection criteria should include:

• Epitope location: Consider antibodies targeting different regions of ELOVL5 depending on your experimental goals. Some antibodies recognize the peptide sequence NNVKPRKLR, which is conserved in primates .

• Experimental application match:

  • For protein-protein interaction studies: Choose antibodies validated for immunoprecipitation

  • For subcellular localization: Select antibodies validated for immunofluorescence

  • For tissue expression analysis: Use IHC-validated antibodies

• Species reactivity: Ensure cross-reactivity with your model system (human, mouse, rat). Some antibodies show cross-reactivity across multiple species, while others are species-specific .

• Validation data: Examine the manufacturer's validation data, including western blot images showing the expected molecular weight (approximately 25-35 kDa) and immunohistochemistry images demonstrating the expected subcellular localization pattern in the endoplasmic reticulum .

What are the key considerations when investigating ELOVL5's role in cancer progression?

When investigating ELOVL5 in cancer:

• Expression analysis: Compare ELOVL5 expression between normal and cancer tissues. In breast cancer, lower ELOVL5 expression has been associated with worse prognosis in ER+ patients and increased metastatic potential .

• Functional assays:

  • Proliferation assays: Downregulation of ELOVL5 has been shown to limit breast cancer cell proliferation

  • Migration/invasion assays: ELOVL5 suppression can promote EMT and cell invasion

  • Lipid droplet analysis: ELOVL5 knockdown induces lipid droplet accumulation, which affects TGF-β receptor expression

• Signaling pathway analysis: In renal cell carcinoma, ELOVL5 affects AKT-mTOR-STAT3 signaling, promoting cellular proliferation and invasion .

• In vivo metastasis models: ELOVL5 suppression has been shown to promote lung metastases in murine breast cancer models .

• Rescue experiments: Supplementation with arachidonic acid and eicosapentaenoic acid (ELOVL5 end products) can partially reverse cellular effects of ELOVL5 knockout .

How can I investigate the relationship between ELOVL5 and cellular signaling pathways?

To study ELOVL5's impact on signaling pathways:

• Phosphorylation analysis: ELOVL5 regulates Akt2 and FoxO1 phosphorylation, particularly at the S473 site of Akt2 and S256 site of FoxO1, but not Akt-T308 phosphorylation .

• Protein complex analysis: Immunoprecipitation studies have shown that elevated ELOVL5 activity increases the interaction between rictor and mTOR, components of the mTORC2 complex .

• Pathway inhibitors: Use specific inhibitors of mTORC1 (rapamycin) versus mTORC2 (rictor siRNA) to distinguish which complex is affected by ELOVL5 activity.

• Fatty acid profiling: Analyze changes in fatty acid profiles in response to ELOVL5 manipulation, focusing on the 18:1,n-7/16:1,n-7 ratio, which has been shown to increase with elevated ELOVL5 activity .

• Downstream target gene expression: Measure expression of genes regulated by the mTORC2-Akt-FOXO1 pathway, such as Pck1, following ELOVL5 manipulation .

What controls should I include when validating ELOVL5 antibody specificity?

For rigorous validation of ELOVL5 antibodies:

• Positive controls:

  • Cell lines: HeLa, MCF-7, HepG2, and Jurkat cells express ELOVL5

  • Tissues: Adrenal gland, testis, liver, and kidney tissues should show ELOVL5 expression

• Negative controls:

  • ELOVL5 knockout/knockdown: CRISPR/Cas9-mediated knockout or siRNA-mediated knockdown cells

  • Secondary antibody-only control: Omit primary antibody to assess background

  • Isotype control: Use non-specific IgG of the same isotype to evaluate non-specific binding

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide (if available) to block specific binding sites

• Cross-validation: Compare results from different antibody clones targeting different epitopes of ELOVL5

• Orthogonal methods: Validate protein expression using complementary techniques such as mass spectrometry or RNA expression analysis

How should I interpret contradictory findings regarding ELOVL5 expression in cancer?

When faced with contradictory findings:

• Consider cancer subtype specificity: ELOVL5 shows different expression patterns in different cancer subtypes. For example, low ELOVL5 expression is associated with worse prognosis specifically in ER+ breast cancer patients , while elevated ELOVL5 is associated with poor prognosis in renal cell carcinoma .

• Analyze stage-dependent effects: ELOVL5 may have different roles in cancer initiation versus progression. In breast cancer, ELOVL5 downregulation limits proliferation but promotes invasion and metastasis .

• Examine context-dependent functions: ELOVL5's role depends on the lipid metabolism context of the specific tissue or cancer type.

• Evaluate methodology differences: Different antibodies, detection methods, and normalization approaches can yield seemingly contradictory results.

• Integrate multiple approaches: Combine protein expression (western blot, IHC) with functional assays and in vivo models to develop a comprehensive understanding of ELOVL5's role.

How can I design experiments to study ELOVL5's role in lipid metabolism regulation?

A comprehensive experimental approach should include:

• Genetic manipulation strategies:

  • CRISPR/Cas9 knockout: For complete elimination of ELOVL5 function

  • siRNA/shRNA: For transient or stable knockdown

  • Overexpression: Using adenoviral vectors (Ad-Elovl5) as described in multiple studies

• Fatty acid profiling:

  • Analyze changes in fatty acid composition using liquid chromatography/electrospray ionization-tandem mass spectrometry

  • Focus on ELOVL5 substrates (18:3,n-6) and products (arachidonic acid, eicosapentaenoic acid)

  • Calculate ratios of product:substrate fatty acids as indicators of ELOVL5 activity

• Rescue experiments:

  • Supplement with specific fatty acids (arachidonic acid, eicosapentaenoic acid) to rescue ELOVL5 deficiency

  • Use structural analogs to determine specificity

• Metabolic pathway analysis:

  • Measure triglyceride content and β-hydroxybutyrate levels

  • Analyze lipid droplet formation using fluorescent dyes like BODIPY or Oil Red O

  • Examine endoplasmic reticulum stress markers, which can be affected by ELOVL5 activity

What approaches can I use to study ELOVL5 phosphorylation and its impact on substrate preference?

To investigate ELOVL5 phosphorylation:

• Phosphorylation site identification:

  • Use phospho-specific antibodies if available

  • Employ mass spectrometry to identify phosphorylation sites

  • Consider in silico prediction of potential phosphorylation sites

• Substrate preference analysis:

  • Design in vitro elongation assays with different fatty acid substrates

  • In an essential fatty acid-deficient state, examine changes in Mead acid (20:3n-9) synthesis, which can be affected by ELOVL5 phosphorylation

  • Compare elongation activity toward different substrates (e.g., 18:1n-9, 18:2n-6, 18:3n-3)

• Kinase identification:

  • Use kinase inhibitors to identify responsible kinases

  • Perform kinase assays with recombinant ELOVL5 protein

• Phosphomimetic mutations:

  • Generate phosphomimetic (e.g., Ser to Asp) and phosphodeficient (e.g., Ser to Ala) mutants

  • Compare substrate preferences of wild-type and mutant ELOVL5

• Physiological triggers:

  • Study how essential fatty acid deficiency affects ELOVL5 phosphorylation status

  • Examine how different PUFA treatments (C18 vs. C20 PUFAs) affect ELOVL5 phosphorylation

What are the solutions for weak or absent ELOVL5 signal in western blotting?

For improving western blot detection:

• Protein extraction optimization:

  • Use stronger lysis buffers with 1% SDS for membrane proteins

  • Include protease inhibitors to prevent degradation

  • Avoid freeze-thaw cycles

• Antibody selection and concentration:

  • Try different antibody clones - some detect ELOVL5 around 25 kDa despite its calculated 35 kDa molecular weight

  • Increase antibody concentration (1:1000 to 1:500)

  • Extended incubation times (overnight at 4°C)

• Sample preparation:

  • Avoid high temperatures during sample preparation

  • Use fresh samples

  • Include reducing agents (β-mercaptoethanol or DTT)

• Detection system:

  • Use more sensitive detection systems (ECL Plus, SuperSignal West Femto)

  • Consider HRP-conjugated antibodies for direct detection

  • Increase exposure times

• Positive controls:

  • Include validated positive controls (HeLa, MCF-7, kidney tissue)

  • Consider recombinant ELOVL5 protein as a standard

How can I optimize immunohistochemistry protocols for ELOVL5 detection in different tissues?

For tissue-specific IHC optimization:

• Fixation considerations:

  • For brain tissue: Brief fixation (24-48 hours) in 4% PFA

  • For liver: Standard 10% neutral buffered formalin

  • For fatty tissues: Longer fixation times may be needed

• Antigen retrieval optimization:

  • Test both heat-induced epitope retrieval methods: citrate buffer (pH 6.0) and TE buffer (pH 9.0)

  • Adjust retrieval times (10-30 minutes)

  • Consider enzymatic retrieval for certain tissues

• Background reduction:

  • Include blocking steps with serum from the same species as the secondary antibody

  • Add 0.1-0.3% Triton X-100 to reduce non-specific binding

  • Consider avidin/biotin blocking for tissues with high endogenous biotin

• Signal amplification:

  • Implement tyramide signal amplification for low-abundance detection

  • Use polymer-based detection systems

  • Consider overnight primary antibody incubation at 4°C

• Counterstaining adjustments:

  • Reduce hematoxylin intensity for better visualization of DAB-positive areas

  • Use specialized counterstains for specific tissue types

What strategies can overcome difficulties in immunoprecipitating ELOVL5 for protein interaction studies?

For successful ELOVL5 immunoprecipitation:

• Lysis buffer optimization:

  • Use NP-40 or digitonin-based buffers to preserve protein-protein interactions

  • Include phosphatase inhibitors to maintain phosphorylation states

  • Adjust salt concentration (150-300 mM NaCl) to optimize stringency

• Antibody selection:

  • Choose antibodies specifically validated for IP, such as the ELOVL5 Antibody (B-3) AC

  • Consider agarose-conjugated antibodies for direct precipitation

• Cross-linking approach:

  • Use DSP or formaldehyde to stabilize transient interactions

  • Optimize cross-linking time to prevent over-cross-linking

• Co-IP partner detection:

  • For mTOR complexes, specifically look for rictor and raptor interactions

  • Use sensitive western blotting techniques for detection

• Negative controls:

  • Include IgG control immunoprecipitations

  • Consider ELOVL5 knockout/knockdown cells as specificity controls

How can ELOVL5 antibodies be applied in cancer biomarker research?

Emerging applications in cancer biomarker research:

• Prognostic marker development:

  • ELOVL5 expression is associated with poor prognosis in ER+ breast cancer patients

  • In renal cell carcinoma, higher ELOVL5 levels correlate with poor clinical prognosis

• Multiplex immunohistochemistry:

  • Combine ELOVL5 antibodies with other markers (hormone receptors, proliferation markers)

  • Develop tissue microarray analysis for high-throughput screening

• Liquid biopsy applications:

  • Explore ELOVL5 detection in circulating tumor cells

  • Investigate ELOVL5 in exosomes as potential biomarkers

• Therapeutic response prediction:

  • Correlate ELOVL5 expression with response to therapies targeting lipid metabolism

  • Investigate combination with TGF-β receptor inhibitors in ELOVL5-low tumors

• Metastatic potential assessment:

  • Evaluate ELOVL5 expression in primary tumors as a predictor of metastatic potential

  • Consider ELOVL5 in conjunction with lipid droplet markers

What are the emerging roles of ELOVL5 in neurological disorders?

New directions in neurological research:

• Spinocerebellar ataxia:

  • ELOVL5 mutations are associated with spinocerebellar ataxia-38 (SCA38)

  • Antibodies can help characterize ELOVL5 expression patterns in neurological tissues

• Neuroinflammation:

  • Study ELOVL5's role in producing polyunsaturated fatty acids that modulate inflammation

  • Examine ELOVL5 expression in microglial cells during inflammatory responses

• Neurodevelopment:

  • Investigate ELOVL5 expression patterns during brain development

  • Correlate with myelination processes and synapse formation

• Neurodegenerative diseases:

  • Explore ELOVL5 expression changes in Alzheimer's, Parkinson's, and other neurodegenerative conditions

  • Correlate with lipid membrane composition alterations

• Brain region specificity:

  • Map ELOVL5 expression across different brain regions

  • Correlate with region-specific lipid profiles

How might single-cell approaches advance our understanding of ELOVL5 biology?

Single-cell methodologies for ELOVL5 research:

• Single-cell immunofluorescence:

  • Combine ELOVL5 antibodies with subcellular markers (ER, lipid droplets)

  • Correlate ELOVL5 expression with cell cycle markers or differentiation markers

• Mass cytometry (CyTOF):

  • Develop metal-conjugated ELOVL5 antibodies for high-dimensional protein analysis

  • Combine with metabolic markers and signaling pathway components

• Spatial transcriptomics integration:

  • Correlate ELOVL5 protein expression with spatial gene expression patterns

  • Map ELOVL5 expression in complex tissues with preserved spatial context

• Single-cell metabolomics:

  • Correlate single-cell ELOVL5 expression with fatty acid profiles

  • Identify cellular heterogeneity in lipid metabolism

• Live-cell imaging:

  • Develop non-disruptive labeling techniques for ELOVL5 in living cells

  • Track dynamic changes in ELOVL5 localization and activity